Abstract

An assay has been developed that allows the identification of
molecules that function as type I IFN antagonists. Using this assay, we
have identified an Ebola virus-encoded inhibitor of the type I IFN
response, the Ebola virus VP35 protein. The assay relies on the
properties of an influenza virus mutant, influenza delNS1 virus, which
lacks the NS1 ORF and, therefore, does not produce the NS1 protein.
When cells are infected with influenza delNS1 virus, large amounts of
type I IFN are produced. As a consequence, influenza delNS1 virus
replicates poorly. However, high-efficiency transient transfection of a
plasmid encoding a protein that interferes with type I IFN-induced
antiviral functions, such as the influenza A virus NS1 protein or the
herpes simplex virus protein ICP34.5, rescues growth of influenza
delNS1 virus. When plasmids expressing individual Ebola virus proteins
were transfected into Madin Darby canine kidney cells, the Ebola virus
VP35 protein enhanced influenza delNS1 virus growth more than 100-fold.
VP35 subsequently was shown to block double-stranded RNA- and
virus-mediated induction of an IFN-stimulated response element reporter
gene and to block double-stranded RNA- and virus-mediated induction of
the IFN-β promoter. The Ebola virus VP35 therefore is likely to
inhibit induction of type I IFN in Ebola virus-infected cells and may
be an important determinant of Ebola virus virulence in
vivo.

Ebola viruses are enveloped,
negative-strand RNA viruses belonging to the family
Filoviridae. These viruses possess genomes of approximately
19 kb and are known to encode eight proteins, the nucleoprotein (NP),
VP35, VP40, glycoprotein (GP), soluble GP, VP30, VP24, and L
(polymerase) proteins (1). Ebola virus infections frequently result in
severe hemorrhagic fever, and epidemics of the Ebola virus, Zaire
subtype have resulted in mortality rates of greater than 80% (1, 2).
The pathologic features and the immune responses characteristic of
fatal and nonfatal human Ebola virus infections have begun to be
characterized (3–5). Additionally, the mechanisms by which Ebola
viruses induce hemorrhage and shock are beginning to be explored.
Recent reports have suggested roles for both immune-mediated pathology
(3) as well as pathology mediated by specific viral proteins.
Membrane-bound GP has been proposed to mediate cytotoxicity in
endothelial cells (4), whereas soluble GP has been proposed to inhibit
early neutrophil activation (5). However, the latter mechanism is
controversial (6). To fully understand the pathogenesis of Ebola virus
infections, it will be important to study further the mechanisms by
which the virus interacts with its host, including the ways in which
the virus subverts the host antiviral response.

One important component of the host antiviral response is the type I
IFN system. Type I IFN is synthesized in response to viral infection;
double-stranded RNA (dsRNA) or viral infection activates latent
transcription factors, including IRF-3 and NF-κB, resulting in the
transcriptional up-regulation of type I IFN, IFN-α, and IFN-β,
genes. Secreted type I IFNs signal through a common receptor,
activating the JAK/STAT signaling pathway. This signaling stimulates
transcription of IFN-sensitive genes, including a number that encode
antiviral proteins, and leads to the induction of an antiviral state.
Among the antiviral proteins induced in response to type I IFN are
dsRNA-dependent protein kinase R (PKR), 2′,5′-oligoadenylate synthetase
(OAS), and the Mx proteins (7–10).

Many viruses have evolved mechanisms to subvert the host IFN response.
For example, the herpes simplex virus (HSV-1) protein ICP34.5
counteracts the PKR-mediated phosphorylation of translation initiation
factor eIF-2α, preventing the establishment of an IFN-induced block
in protein synthesis (11). In the negative-strand RNA viruses, several
different anti-IFN mechanisms have been identified (12, 13). First, the
influenza A virus NS1 protein was shown to block IFN responses in
virus-infected cells (12). Subsequently, the V protein of SV5 was shown
to target STAT1 for proteasome-mediated degradation, preventing
signaling from both type I and type II IFN receptors (13, 14). Also,
the Sendai virus C proteins were found to block types I and II IFN
signaling and to counteract the establishment of an antiviral state
(15–17). Recently, measles virus infection has been shown to block
induction of type I IFN production (18). Also, the bovine respiratory
syncytial virus NS1 and NS2 proteins have been shown to function
together to antagonize the type I IFN response (51).

The best-studied example of an IFN antagonist encoded by a
negative-strand RNA virus is the influenza A virus NS1 protein. A
mutant influenza virus, influenza delNS1 virus, which lacks the NS1 ORF
and, therefore, produces no NS1 protein, grows poorly on substrates in
which type I IFN-induced antiviral pathways are intact (12). Such
substrates include Madin Darby canine kidney (MDCK) cells, 10-day-old
embryonated chicken eggs, and mice. It is clear that the growth of
influenza delNS1 virus is impaired because of its inability to
counteract IFN-mediated antiviral response(s). The virus grows
similarly to wild-type virus [influenza A/PR/8/34 (H1N1) (PR8)
virus] on substrates such as 6-day-old embryonated chicken eggs, Vero
cells, and STAT1−/− mice, which do not mount
an effective type I IFN response (12, 19). The failure of influenza
delNS1 virus to grow on IFN-producing substrates correlates with its
ability to induce IFN. Thus, infection of cells with this mutant virus
induces large amounts of type I IFN, a circumstance associated with a
potent antiviral state (ref. 19; M. Salvatore, H. Zheng, T. Muster,
P.P., and A.G.-S., unpublished results). Such IFN synthesis does not
occur when the same substrates are infected with the NS1
protein-producing wild-type PR8 virus (12). The ability of the NS1
protein to prevent IFN production and to facilitate growth of influenza
viruses on IFN-producing substrates may be related to its ability to
bind single-stranded and/or dsRNA (20–22). NS1 has been shown to
inhibit activation of PKR and OAS (21). Additionally, the NS1 protein
is able to prevent viral activation of IRF-3 (23) and NF-κB (52),
central components in promoting type I IFN synthesis.

Ebola virus-infected endothelial cells have impaired IFN responses,
suggesting that Ebola virus also may encode an IFN antagonist. After
treatment with dsRNA, IFN-α, or IFN-γ, little induction of
IFN-stimulated genes was seen in infected cells compared with
uninfected cells (24, 25). However, this does not reflect a general
inhibition of signal transduction; IL-1β-induced signaling was not
inhibited in Ebola virus-infected cells (24, 25).

In this report, we describe an assay that permits identification of
proteins that inhibit the type I IFN-induced antiviral response. This
assay was used further to screen for an Ebola virus-encoded IFN
antagonist. The assay uses transient transfection of plasmids encoding
type I IFN antagonists to enhance growth on MDCK cells of the
IFN-sensitive influenza delNS1 virus. Thus, expression of the influenza
A virus NS1 protein or the HSV-1 ICP34.5 efficiently complemented
growth of influenza delNS1 virus. Using this assay, we screened
individual Ebola virus proteins for their ability to rescue growth of
influenza delNS1 virus on MDCK cells. One Ebola virus protein, the VP35
protein, was found to complement influenza delNS1 virus growth. VP35
subsequently was found to block dsRNA- and virus-mediated induction of
an IFN-responsive promoter and the IFN-β promoter. Therefore, the
VP35 protein is likely to function as an inhibitor of the type I IFN
response in Ebola virus-infected cells and may be an important
determinant of Ebola virus virulence.

Plasmids.

The NS1 expression plasmid pCAGGS-PR8 NS1 SAM has been described (23).
The NS1 expression plasmid pCMV-PR8 NS1 SAM was generated by subcloning
the NS1 ORF from pCAGGS-PR8 NS1 SAM into pcDNA3 (Invitrogen). The HSV-1
γ134.5 gene was excised with NcoI
and BamHI from the plasmid pBR4789 (a generous gift from B.
Roizman, University of Chicago), which contains the HSV-1
BamHI S1 fragment, and subcloned into pCAGGS (26).

The Ebola virus strain Mayinga, subtype Zaire, was used for cloning
(nucleotide numbers refer to the Ebola virus Zaire genome, GenBank
accession number AF086833). The ORFs and small parts of the
nontranslated regions of the NP, VP35, and VP30 genes were amplified by
reverse transcription—PCR, with specific primers containing
appropriate restriction sites. The products were inserted into the
expression vector pcDNA3. NP was inserted by using BamHI.
VP35 (nucleotides 3,126–4,175) was inserted by using BamHI
and NotI. The VP30 gene (nucleotides 8,506–9,399) was
inserted by using EcoRI and XhoI. The GP and
soluble GP inserts were excised from the plasmids pGEM-mGP7 and
pGEM-mGP8 (27) with BamHI and HindIII. The
HindIII sites were blunted, and the fragments were inserted
between the BamHI–EcoRV restriction sites of
pcDNA3.1(+) (Invitrogen). RNA-cDNA hybrids from a cDNA library of the
Ebola virus subtype Zaire, strain Mayinga, (28) were used to
amplify the VP40 and VP24 genes. PCR fragments containing ORFs of
VP40 (nucleotides 4,410–5,779) and VP24 (nucleotides 10,290–11,312)
genes were inserted at the EcoRV restriction site of
pcDNA3.1(+). The clones were confirmed by DNA sequencing.

Influenza delNS1 Virus Complementation Assays.

High-efficiency transient transfection of MDCK cells was performed by
using Lipofectamine2000 (LF2000) (GIBCO/BRL). Four micrograms of the
indicated expression plasmid was adjusted to 50 μl by using Optimem I
medium (GIBCO/BRL). Per transfection, 10 μl of LF2000 was adjusted
to 0.25 ml with Optimem I medium and incubated in a 5-ml polystyrene
snap-cap tube at room temperature for 5 min. Each 50-μl DNA sample
was added to the 0.25-ml LF2000/Optimem I mix, agitated gently, and
incubated 20 min at room temperature. A confluent
80-cm2 flask of MDCK cells was detached with
trypsin. The cells were brought to 12 ml with DMEM/10% FBS (no
antibiotics), pelleted at 1,000 rpm for 5 min in a table-top
centrifuge, and, after aspiration of the supernatant, resuspended in
DMEM/10% FBS (no antibiotics) to a concentration of 4 ×
106 cells/ml. A portion (0.25 ml) of the cell
suspension was aliquoted into 35-mm tissue culture dishes. After the
20-min incubation period, 1 ml of DMEM/10% FBS (no antibiotics) was
added to each DNA/LF2000 mix, and the DNA/LF2000/medium mixture
was added to the dishes containing the MDCK cells. After mixing, the
cells were maintained at 37°C overnight. Sixteen to 20 h
posttransfection, the cells were infected with
103 plaque-forming units (pfu) of influenza
delNS1 virus [multiplicity of infection (moi) = 0.001] in a
volume of 0.1 ml. After removal of the inoculum, the cells were
maintained in 1.5 ml of DMEM/0.3% bovine albumin/3 μg/ml trypsin
(trypsin, 1:250; Difco).

Twenty-four hours posttransfection, the cells were mock-treated,
transfected with 40 μg polyI:polyC (Amersham Pharmacia) by using
LF2000 according to the manufacturer's recommendations, or infected
with influenza delNS1 virus or Sendai virus at an moi of 1.0 for each
virus. Cells treated by all methods were maintained in DMEM/0.3%
bovine albumin. Twenty-four hours posttreatment, the cells were
harvested and lysed in Reporter Lysis Buffer (Promega). CAT assays were
performed as described (31). Luciferase assays were performed by using
the Promega luciferase assay system according to the manufacturer's
directions.

Western Blot Analysis.

293 cells were transfected with 4 μg of the indicated plasmids by
using LF2000, as described above. Forty-eight hours posttransfection,
the cells were lysed in SDS/PAGE sample buffer, and Western blots
were performed by following standard procedures. The influenza A virus
NS1 protein was detected by using anti-PR8 NS1 rabbit polyclonal
antiserum 5091 at a 1:1,000 dilution. The Ebola virus NP and VP35
proteins were detected by using a goat polyclonal anti-Ebola virus
antiserum (purchased from Vector Laboratories) at a 1:20,000 dilution.
Secondary antibodies were anti-rabbit or anti-goat antibodies
conjugated to horseradish peroxidase. Detection was performed by using
the NEN Renaissance Western blot chemiluminescence reagent.

Northern Blot Analysis.

293 cells were transfected with 5 μg of either empty vector or the
VP35 expression plasmid by using LF2000. Twenty-four hours
posttransfection, the cells were infected with influenza delNS1 virus
or Sendai virus at an moi of 1. Twenty-four hours later, total RNA was
extracted by using TRI reagent (Molecular Research Center, Cincinnati).
Ten micrograms of total RNA was used for Northern blot analysis by
using Quickhyb hybridization solution (Stratagene). IFN-β or
β-actin mRNAs were detected by hybridization to specific
[32P]ATP-labeled probes.

Results

The influenza delNS1 virus grows poorly on IFN-producing cells such as
MDCK cells. However, wild-type PR8 virus, isogenic with influenza
delNS1 virus except that it produces the NS1 protein, grows to high
titer on MDCK cells (12). It therefore was determined whether
high-efficiency transfection of MDCK cells with an NS1-expression
plasmid would complement growth of influenza delNS1 virus (Fig.
1). When MDCK cells were transfected with
an influenza A virus NS1 plasmid and infected 24 h later with
influenza delNS1 virus at a low moi, the mutant virus grew to
approximately 1 × 106 pfu/ml. However,
infection of cells transfected with an empty plasmid yielded
102 pfu/ml or less (Fig. 1).

Growth of the influenza delNS1 virus is complemented by transient
transfection of an influenza A NS1 protein or an HSV ICP34.5 expression
plasmid. MDCK cells were transfected with 4 μg of empty expression
plasmid (pCAGGS), pCAGGS-PR8 NS1 SAM (23), or
pCAGGS-γ134.5. Twenty-four hours later, the cells were
infected with influenza delNS1 virus (moi = 0.001). Forty-eight
hours posttransfection, viral titers were determined by plaque assay.
The results are the average of two independent experiments.

It then was determined whether expression of another known inhibitor of
the type I IFN-induced antiviral response, HSV-1 ICP34.5, would
complement growth of influenza delNS1 virus. Expression of the
HSV-1-encoded PKR antagonist ICP34.5 (11) clearly complemented growth
of the influenza delNS1 virus (Fig. 1). This result suggests that
complementation of influenza delNS1 virus growth reflects an anti-IFN
function. It also indicates that this complementation assay can be used
to identify other proteins that inhibit the IFN-induced antiviral
response.

Expression of the Ebola Virus VP35 Protein Blocks Induction of an
ISRE Promoter.

To determine whether VP35 inhibits the dsRNA- and virus-mediated
activation of IFN-sensitive gene expression, cells were transfected
with an ISRE-driven CAT-reporter plasmid and a constitutively
expressed, simian virus 40 promoter-driven luciferase reporter plasmid.
Additionally, the cells were transfected with empty vector, NS1
expression plasmid, VP35 expression plasmid, or, as an additional
control, an Ebola virus NP expression plasmid. One day later, the cells
were mock-treated, transfected with dsRNA, or infected with either
influenza delNS1 virus or with Sendai virus, strain Cantell (an
attenuated strain known to induce large amounts of IFN). After an
additional 24 h, cell lysates were prepared and assayed for CAT
activity and luciferase activity (Fig.
3A). Transfection of cells
with dsRNA or infection with either influenza delNS1 virus or Sendai
virus gave a strong induction of the IFN-sensitive promoter. When
either NS1 or VP35 was present, expression from the IFN-responsive
promoter was almost completely blocked. Levels of ISRE induction,
normalized to levels of luciferase activity, are shown in Fig.
3A. Expression of the control luciferase reporter plasmid
was not inhibited by expression of either NS1 or VP35 (data not shown).
Expression of the Ebola virus NP, which did not complement growth of
influenza delNS1 virus, did not inhibit activation of the ISRE promoter
(data not shown). Expression of the NS1, VP35, and NP proteins was
confirmed by Western blotting (Fig. 3B). These results show
that both NS1 and VP35 can block type I IFN production and/or
signaling in response to either dsRNA treatment or to viral infection.

Expression of Ebola virus VP35 protein inhibits dsRNA- or
virus-mediated induction of an ISRE. (A) Fold induction
of an ISRE promoter–CAT reporter gene in the presence of empty vector,
NS1 expression plasmid, or VP35 expression plasmid. 293 cells were
transfected with 4 μg of the indicated expression plasmid plus 0.3
μg each of the reporter plasmids pHISG-54-CAT and pGL2-Control.
Twenty-four hours posttransfection, the cells were mock-treated or
treated with the indicated IFN inducer. The CAT activities were
normalized to the corresponding luciferase activities to determine fold
induction. (B) Western blot showing NS1, VP35, and Ebola
virus NP expression. 293 cells were transfected with 4 μg of the
indicated plasmids. Forty-eight hours later, cell lysates were prepared
and Western blots were performed by using the indicated antiserum.

Expression of the Ebola Virus VP35 Protein Blocks Activation of the
INF-β Promoter.

In wild-type influenza A virus-infected cells, the NS1 protein blocks
induction of type I IFN. This block is due, in large part, to the
ability of NS1 to prevent activation of IRF-3 (23) and NF-κB (52),
two transcription factors that play a critical role in stimulating the
synthesis of IFN-β. Synthesis of IFN-β, in turn, plays an important
role in the initiation of the type I IFN cascade (32). The Ebola virus
VP35, therefore, was tested for its ability to block activation of the
IFN-β promoter.

Empty vector, NS1 expression plasmid, or VP35 expression plasmid was
cotransfected with a mouse IFN-β promoter-driven CAT reporter and a
simian virus 40 promoter-driven luciferase reporter. When cells
subsequently were transfected with dsRNA, a strong induction of the
IFN-β promoter was observed in empty vector-transfected cells, but
this induction was blocked when either NS1 or VP35 was expressed (Fig.
4A).

The VP35 protein of Ebola virus inhibits induction of the IFN-β
promoter. (A) Inhibition of induction of the mouse
IFN-β promoter. 293 cells were transfected with 4 μg of the
indicated expression plasmid plus 0.3 μg each of the reporter
plasmids pIFN-β-CAT and pGL2-Control. Twenty-four hours
posttransfection, the cells were mock-transfected or transfected with
40 μg of polyI:polyC. (B) Northern blot showing
VP35-mediated inhibition of endogenous IFN-β induction.
293 cells were transfected with either empty vector or VP35 expression
plasmid. Twenty-four hours later, the cells were mock-infected (−) or
infected with influenza delNS1 virus (delNS1) or Sendai virus (SeV)
(moi = 1). Total RNA was prepared from cells at 10 h or
20 h posttransfection. Mock-transfected cell RNA was prepared at
the same time as the 20-h postinfection samples. Northern blots were
performed to detect IFN-β or β-actin mRNAs. Note that less total
RNA was obtained when cells, including the mock-infected cells, were
lysed at the 20-h postinfection time point.

It also was determined whether VP35 could block activation of the
endogenous human IFN-β promoter. Cells were transfected
with empty vector or VP35 expression plasmid and, 24 h later,
mock-infected or infected with influenza delNS1 virus or with Sendai
virus. Ten or 20 h postinfection, total cellular RNA was isolated,
and a Northern blot was performed to detect IFN-β mRNA (Fig.
4B). Expression of VP35 clearly blocked induction of the
endogenous IFN-β promoter. Before infection with either
virus, IFN-β mRNA was undetectable. After infection, when the IFN-β
mRNA levels were normalized to β-actin mRNA levels, it was found
that, in influenza delNS1 virus-infected cells, the presence of VP35
reduced IFN-β induction 8-fold at 10 h postinfection and
8.4-fold at 20 h posttransfection. In Sendai virus-infected cells,
the presence of VP35 reduced IFN-β induction 6.1-fold at 10 h
posttransfection and 5.9-fold at 20 h posttransfection.

The VP35 protein is an essential component of the Ebola virus RNA
synthesis complex and likely associates with the viral NP (33, 34).
Therefore, it was determined whether Ebola virus VP35 retained its
IFN-antagonizing properties when it was coexpressed with the Ebola
virus NP. An ISRE-reporter assay was performed in which cells received
either empty vector, VP35 alone, NP alone, or a combination of VP35 and
NP. Twenty-four hours posttransfection, the cells were transfected with
dsRNA or infected with Sendai virus. As seen previously, transfection
with empty plasmid or with NP expression plasmid did not block
activation of the ISRE promoter, but expression of VP35 did block its
activation (Fig. 5). Further,
coexpression of VP35 and NP was able to block ISRE activation to the
same extent as expression of VP35 alone (Fig. 5). These data indicate
that VP35, even when coexpressed with the Ebola virus NP, can act as an
IFN antagonist.

The Ebola virus VP35 protein inhibits type I IFN induction when
coexpressed with Ebola virus NP. Fold induction of the IFN-inducible
ISRE-driven reporter in the presence of empty vector, VP35, NP, or VP35
plus NP. 293 cells were transfected with a total of 4 μg of
expression plasmid, including 2 μg of a plasmid encoding an
individual protein and 2 μg of a second plasmid (either empty vector
or a second expression plasmid) plus 0.3 μg each of the reporter
plasmids pHISG-54-CAT and pGL2-Control. Twenty-four hours
posttransfection, the cells were mock-treated or treated with the
indicated IFN inducer. Twenty-four hours postinduction, CAT and
luciferase assays were performed. The CAT activities were normalized to
the corresponding luciferase activities to determine fold induction.

Discussion

In this report, the Ebola virus VP35 protein was identified as an
IFN antagonist based on its ability to complement growth of the
influenza delNS1 virus. VP35 shares this complementing ability with the
influenza A virus NS1 protein, the HSV-1 ICP34.5 (Fig. 1), and the
vaccinia virus E3L protein (unpublished observation). Each of these
proteins has been shown to interfere with one or more components of the
IFN-induced antiviral response. The NS1 protein of influenza A virus
blocks both the production of type I IFN (12) and the activation of the
IFN-induced antiviral proteins PKR (21, 35, 36) and OAS (unpublished
observation). The inhibition of type I IFN production occurs, at least
in part, because NS1 blocks activation of the latent transcription
factors IRF-3 and NF-κB (23, 52), which are involved in the dsRNA-
and virus-mediated activation of the IFN-β promoter. These inhibitory
functions may be mediated by the binding of dsRNA by NS1 (21, 23, 52).
The vaccinia virus E3L protein also blocks the activation of both PKR
(37–40) and OAS (41). Further, E3L expression promotes growth of
vaccinia virus in the presence of IFN (40, 42). The HSV-1 ICP34.5
antagonizes the type I IFN antiviral response by targeting
phosphorylated eIF-2α, the product of activated PKR. Specifically,
ICP34.5 binds the cellular protein phosphatase 1α and retargets it to
eIF-2α (11). The resulting dephosphorylation of eIF-2α thus
counteracts PKR function (11). As a result, virus mutants that fail to
make ICP34.5 are attenuated and show restricted tissue tropism in mice
(43, 44). However, as with the influenza delNS1 virus, virulence of
these mutants is restored in mice lacking genes critical to type I IFN
signaling or in mice lacking PKR (45, 46). Because the HSV-1 ICP34.5
rescues influenza delNS1 virus growth, a VP35-mediated block of PKR
function likely would be sufficient to enhance influenza delNS1 virus
growth. However, any number of mechanisms might accomplish this task,
including a direct effect on PKR function, a block in type I
IFN-induced signaling, such that IFN-responsive genes (including PKR)
are not transcriptionally activated, or a block in IFN synthesis. We
also demonstrated that VP35, like the influenza A virus NS1 protein,
prevents dsRNA- and virus-mediated activation of ISRE-containing
promoters (Fig. 3) and the IFN-β promoter (Fig. 4). In blocking
virus-induced production of IFN, VP35 would prevent the establishment
of an antiviral state in both uninfected cells and Ebola virus-infected
cells and, thus, facilitate viral replication.

Previously, VP35 has been shown to play an essential role in Ebola
virus RNA synthesis (33). VP35 appears to be an ortholog of the
paramyxovirus and rhabodvirus P proteins (phosphoproteins) (33),
although the filovirus VP35 proteins are only weakly phosphorylated
(47). The IFN antagonists of two other nonsegmented negative-strand
viruses, the SV5 V protein and Sendai virus C proteins, also are
encoded within P genes. However, these paramyxovirus proteins are not
equivalent to P proteins. The Sendai virus C proteins are encoded by
overlapping ORFs that initiate at alternative start codons and are read
from an alternate reading frame (48). The SV5 V protein shares a common
amino terminus with the P protein; however, because of the insertion by
the viral polymerase of a nontemplate encoded G residue, the V protein
possesses a carboxyl terminus distinct from that of the P protein.
There is no evidence for production of either C or V orthologs from the
Ebola virus VP35 gene. There are no ORFs of significant length near the
5′ end of the VP35 gene that would encode a C protein. Additionally,
although mRNA editing by the Ebola virus polymerase occurs within the
GP gene (27, 49), there is no evidence for the presence of RNA-editing
signals within the VP35 gene, nor is there evidence for production of
an edited VP35 gene product. Given the results of our study, the P
proteins of other nonsegmented negative-strand viruses also should be
examined for IFN-antagonizing capability.

The production of an IFN antagonist may contribute to the virulence of
Ebola viruses. In humans, it appears that an appropriate cytokine
response is related to the development of asymptomatic or nonfatal
Ebola virus infection (50). Thus, a viral factor that influences type I
IFN production might influence viral pathology. It is clear from the
study of other viruses that the presence of an IFN antagonist is
required for full virulence. For example, wild-type influenza
A/PR/8/34 virus is pathogenic in wild-type mice
(LD50 =
102–103 pfu), but the
influenza delNS1 virus is severely attenuated
(LD50 > 106 PFU). However,
when the IFN-induced antiviral response is absent, as in
STAT1−/− mice, influenza delNS1 virus is
nearly as virulent as wild-type virus (12). Additionally, influenza
viruses with truncated NS1 proteins display diminished capacity to
replicate in wild-type mice (19). Likewise, HSV-1 mutants that do not
produce ICP34.5 are attenuated in wild-type mice but not in IFN
receptor−/− mice or
PKR−/− mice (45, 46). To experimentally
assess the significance of the VP35 IFN-antagonizing function on Ebola
virus virulence, it ultimately will be necessary to generate viral
mutants that retain the ability to replicate their genomes but that
lack the VP35-encoded IFN-inhibiting function. The development of an
Ebola virus reverse genetics system, toward which significant progress
has been made (33), will facilitate such analyses. It also will be of
interest to assess the relative IFN-inhibiting potency of VP35 proteins
from Ebola virus strains that have displayed different levels of
pathogenicity in humans.

The influenza delNS1 complementation assay described in this manuscript
provides a straightforward way in which to screen proteins for
IFN-antagonizing function. It should be possible, using this system, to
identify the proteins of other viruses that, like the Ebola virus VP35
protein, function as IFN antagonists. The identification of novel,
viral-encoded IFN antagonists will further our understanding of how
pathogens evade the innate immune response. Further, mutation of
viral-encoded IFN antagonists may be an ideal way in which to generate
stable, attenuated live vaccines for a variety of different viral
pathogens. Such an approach already is being investigated for influenza
viruses (19). Viral-encoded IFN antagonists also may become targets for
new antiviral drugs against important human pathogens. Inhibition of
the activity of viral IFN antagonists should result in an induction of
antiviral pathways in the infected cell and a concomitant inhibition of
virus replication.

Acknowledgments

We thank Louis Nguyenvu for excellent technical assistance. This
work was supported by a National Institutes of Health National Research
Service Award postdoctoral fellowship to C.F.B., by research grants
from the National Institutes of Health to A.-G.S. and P.P. and from the
Deutsche Forschungsgemeinschaft to E.M. and H.-D.K.

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